with rigor kinesin [K350, residues 1 to 350,
E236A mutation (K350E236A)] and MAP7
(Fig. 3C). Because distinct binding sites of
MAP7 and kinesin can be artifactually averaged
during reconstruction (fig. S9B), we performed
focused 3D classification around the putative
shared binding site of MAP7 and kinesin (fig. S10;
see methods). The classification resulted in two
distinct maps of the binding site, one occupied by
MAP7 only, and the other occupied by kinesin
only (Fig. 3D). Thus, kinesin and MAP7 cannot
simultaneously bind to the same tubulin dimer.
Consistent with cryo-EM, MTBD was unable
to bind MTs predecorated with K350E236A
(Fig.3E).However,FLMAP7oraconstruct
lacking the MTBD (DMTBD; fig. S11, A to C)
still bound to K350E236A-decorated MTs, pre-
sumably through the P123 domain (residues
175 to 316) ( 8 , 9 ). MT binding ofDMTBD was
nearly abolished upon cleaving the flexible
tails of tubulin (Fig. 3E and fig. S11D), ex-
plaining why P123 was invisible in our struc-
ture. To verify that kinesin is inhibited at high
levels of MAP7 decoration as a result of bind-
ing site overlap, we also replaced the MT-
binding regions of MAP7 with that of tau,
which also overlaps with the tubulin bind-
ing site of kinesin and inhibits kinesin motility(fig. S12) ( 13 , 22 ). Similar to FL MAP7, bind-
ing of this chimeric MAP to the MT resulted
in biphasic regulation of kinesin motility
(Fig.3,FandG).
To reveal how kinesin“walks”along MAP7-
decorated MTs despite competing for the same
binding site, we fluorescently labeled the motor
domain of K560 and tracked kinesin stepping
with nanometer precision under limiting aden-
osine triphosphate (ATP) conditions (Fig. 4A).
On undecorated MTs, kinesin took 16-nm steps
in the forward direction ( 23 ), whereas steps
in the sideways and backward directions
were rare (9 and 3%, respectively; Fig. 4, ASCIENCEscience.org 21 JANUARY 2022•VOL 375 ISSUE 6578 329
Fig. 3. MT tethering enables kinesin to move on MAP7-decorated MTs despite
their overlapping binding sites.(A) Schematic of kinesin, MAP7, and tau
constructs. (B) Kymographs showing kinesin motility in the presence of FL and
truncated MAP7. (C) Average cryo-EM map of an MT decorated with both MAP7 and
rigor kinesin. Atomic model of MAP7 and previously reported structure of kinesin
on MT (PDB code: 4HNA) ( 20 ) were fitted into the cryo-EM map. (D) Focused 3D
classification resulted in two distinct density maps showing either MAP7-bound
(left) or kinesin-bound (right) tubulin, indicating competitive binding. (E) Fluorescent
signal (top) and normalized intensity (bottom) of 1mM LD655-labeled MAP7
constructs on MTs that are either untreated (control), subtilisin-treated, or
predecorated with 1mM rigor kinesin (mean ± SEM;nvalues are given for each bar).
(F) Kinesin motility in the presence of chimeric MAPs. (G) Run frequency and
velocity of kinesin in the presence of Tau-MTBD/MAP7-projection (mean ± SEM,
n= 27, 112, 211, 102, and 55 from left to right, two technical replicates).RESEARCH | REPORTS